PROJECT SUMMARY It is difficult to overstate the impact Yersinia pestis, the bacterial agent of plague, has had on human history. It is of great concern as a potential agent of bioterrorism because of its highly virulent nature, and it consistently infects thousands of people per year in endemic foci around the world, including the southwestern United States. Despite these facts, there is much that is unknown about the ability of Y. pestis to infect both flea vectors and mammalian hosts, and its ability to undergo successful transmission between them. The goal of this project is to determine the genetic and molecular mechanisms of resistance to cationic antimicrobial peptides (CAMPs) in Y. pestis, and the role this resistance plays in allowing it to successfully infect fleas. Data from our lab revealed that numerous Y. pestis transposon mutants that were unable to maintain successful flea infections also showed in vitro hypersensitivity to CAMPs. Two mutants contained insertions in genes predicted to block modification of the outer membrane lipopolysaccharide, and we recently published data that this was in fact the cause of the mutants' CAMP sensitivity. Several mutations however were in novel or hypothetical genes not previously known to be involved in CAMP resistance. Additionally, disruption of genes involved in biosynthesis of the enterobacterial common antigen resulted in unexpected CAMP-susceptibility in Y. pestis by an unknown mechanism. These data, along with the recent discovery of the plasmid-mediated mcr colistin resistance genes, indicate that CAMP resistance is more complex, and less well understood, than previously thought. The first specific aim of this project is to investigate structural and genetic changes present in these CAMP- susceptible mutants to improve our understanding of the mechanisms of CAMP resistance. Since CAMPs, such as polymyxins, are often one of the only classes of antibiotics with activity against multi-drug resistant bacterial infections, identifying novel mechanisms of resistance is of critical importance. The second specific aim involves constructing a comprehensive, ordered transposon mutant library of all non-essential genes in Y. pestis KIM6+. This library will be screened for mutants that are susceptible to structurally diverse CAMPs in order to identify additional and potentially novel mechanisms of resistance. Mutants identified in this screen will be analyzed using the biophysical and genetic approaches from the first aim to elucidate their mechanisms of CAMP resistance. The third specific aim is to determine the potential role of different mechanisms of CAMP resistance in transmission by testing the ability of the mutants to maintain infection, and by determining the distribution of mutants in a natural flea vector. The knowledge obtained from this project on CAMP resistance mechanisms will open up a number of new trajectories in plague and antimicrobial resistance research, and the ordered mutant library could stimulate additional investigation into the biology of this important human pathogen. Improving our understanding of CAMP resistance is crucial given the status of polymyxins as WHO ?Critically Important Antimicrobials,? and the recent discovery of transmissible (mcr-1) resistance. Loss of this class of antibiotics could herald the era of untreatable infections. !